Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase ‘Hyd-2’ from Escherichia coli

The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive.


SDS-PAGE of R479K
The electrocatalytic fingerprints of Hyd-2 and R479K Figure S2: Cyclic voltammograms under 100% H 2 for Hyd-2 enzymes. Hyd-2 is shown in black and R479K is in red. While both current maximums for the two scans have been deliberately made comparable (see methods), the shape of the two voltammograms vary widely. Conditions: pH 6.0, 30 o C, 5 mV/s scan rate, 100% H 2 at 1000 scc min -1 , ω= 3000 rpm.
Demonstrating K M H 2 and K i H 2 Figure S3: Varying the concentration of H 2 for the determination of K M (H 2 ) is exemplified here by the R479K variant. Increasing H 2 oxidation activity corresponds with increasing H 2 concentration and the zero-current potential corresponds well to the values predicted by the Nernst equation (inset). For proton reduction the activity increases with decreasing H 2 concentration, indicative of product inhibition, but to a lesser degree than that seen with native enzyme (see text). Other conditions: scan rate 5 mV/s, 30 oC, pH 6, Ar carrier gas, total gas flow rate 1000 scc min-1, ω = 3000 rpm.  Stability of oxidised inactive state(s) Figure S6: Complete anaerobic inactivation of Hyd-2 R479K for determining the stability of oxidised inactive states. The enzyme is fully inactivated in 100% Ar at pH 8 by holding the electrode at approx. +0.36 V. The gas is then switched to 100% H 2 and simultaneously the buffer is exchanged to pH 6 by at least 10-fold volume exchange. After approx. 300 sec to allow for full gas and temperature equilibration of the pH 6 buffer, the cyclic voltammogram from high to low potential is started at 0.1 mV/s (see Figure 7). Other conditions: 30 °C, total gas flow rate 1000 scc min -1 , ω = 3000 rpm. Figure S7: The first derivative of the backwards scan ( Figure 7) for R479K. The asterisked minimum represents the strong inflection point used to denote E switch 3,4 and the arrow indicates a second minimum close to the typical potential of E switch for Hyd-2. Data have been smoothed with the Savitzky-Golay method, 41 points per window, using Origin software.
Response of R479K to transient exposure to O 2 Figure S8: Consecutive cyclic voltammograms of R479K after O 2 exposure. The R479K variant was exposed to O 2 to an initial concentration of 130 µM within a constant H 2 flow. The first scan during which O 2 was injected at 0 V is in black. After the scan was complete a second scan was performed (red). Conditions: 30 °C, pH 6, scan rate 0.5 mV/s, ω=3000 rpm. Scans have not been corrected for film loss.
The temperature dependence of H 2 oxidation and H + reduction current for Hyd-2 and R479K Figure S9: Cyclic voltammograms at varying temperatures for Hyd-2, (A) and R479K (B-D). The relative rate of H 2 oxidation does not track with increasing temperature above 30 o C (C) whereas the relative rate of H + reduction increases with temperature across the temperature range (D). For panels C and D, to increase film stability the film of R479K was prepared using a standard PGE electrode coated with multi-walled carbon nanotubes and pyrene-butyric acid for covalent attachment of the enzyme via its surface lysine residues as described previously. [5][6][7] Conditions: pH 6, scan rate 5 mV/s, 100% H 2 , ω = 3000 rpm. These scans are not corrected for film loss.

Determination of the activation entropy difference (S ‡ ) between Hyd-2 and R479K
The difference in the entropy of activation of Hyd-2 (S 1 ‡ ) and R479K (S 2 ‡ ) was calculated from the ratio of the turnover rates (k 1 and k 2 , respectively) determined by conventional solution assay, and the enthalpies of activation (H 1 ‡ and H 2 ‡ , respectively) determined by PFE ( Table 1). The examples given below are for the lower steady-state rate obtained for R479K in both H 2 oxidation and H + reduction assays as given in Table 1.
multiplying through by R and T gives: this rearranges to give: The value for S ‡ can also be found by calculating the entropy of activation for Hyd-2 (S 1 ‡ ):  The difference in the entropy of activation between Hyd-2 and R479K is then: multiplying through by R and T gives: this rearranges to give: The value for S ‡ can also be found by calculating the entropy of activation for Hyd-2 (S 1 ‡ ): and for R479K (S 2 ‡ ): The difference in the entropy of activation between Hyd-2 and R479K is then:  Electron density distribution across diatomic ligand Figure S12: Electron density distribution across the diatomic ligand with 2Fo-Fc density maps contoured at increasing sigma levels (rmsd values for the electron density map) to highlight the symmetric distribution of electron density in the As-isolated and oxygen treated enzyme compared to the asymmetric distribution in CO treated enzyme. Numerical values for the electron density are summarised in Table S4. Table S4: Absolute peak electron density values (and corresponding map rmsd) for each of the atoms in diatomic ligands bound at the active site indicate symmetric and asymmetric distribution of electron density across the X-Y and CO ligands respectively. Values for the endogenous CO ligand of the Fe atom are added for comparison. The higher density values for the endogenous CO are reflective of the higher occupancy of these atoms.

Enzyme treatment
Atom Peak electron density (e/Å 3 ) Modelling the diatomic ligand in the active site The as-Isolated enzyme shows the diatomic ligand coordinating the catalytic nickel atom. It is stabilised by hydrogen bonds to the side chain of K479 and an ordered water molecule (water '2' in Figure 1C). (B) The reduced enzyme replaces the oxygen species with a loosely coordinated water molecule (water '3' in Figure 1C), which forms two further hydrogen bonds to K479 and the same ordered water as the As-Isolated structure. After treatment of the enzyme with O 2 (C) or CO (D) the bound gas molecules coordinate the Ni atom and are further stabilised by interactions similar to those observed in the As-Isolated enzyme

X-Y
Temperature factors of atoms in the Active site  Transmission IR spectra of R479K in the νCN region Figure S16: Cyanide region of the solution-based transmission IR spectra of anaerobically oxidised R479K (A) Raw data of R479K that has been H 2 -reduced then anaerobically oxidised with DCIP corresponding to the data from Figure 13 (raw data corresponding to Figure 13E, oxidised with DCIP under strict anaerobic conditions after being H 2 -reduced) is shown in black, raw data corresponding to Figure 13F (H 2 -reduced, left overnight and then oxidised with DCIP prepared the previous day) is shown in red, and (B) the 2nd derivatives of the same data. It can be clearly seen that there are no distinguishable IR bands in the high wavenumber region beyond 2112 cm -1 .
IR spectra of poised R479K crystals before and after CO exposure Crystals of R479K Hyd-2 were electrochemically poised in crystallisation buffer containing redox mediators. 8 IR spectra were recorded in an anaerobic glove box using an IR microscope in a transmission cell between CaF 2 windows.
Although the IR spectra for crystals of R479K are difficult to assign on the basis of known, IR-characterised states of native Hyd-2, 9,10 or other native [NiFe]-hydrogenases, 11 it is clear that CO exposure to crystals of R479K at reducing potentials (-700 mV, Figure S17A) does not give rise to the 'high wavenumber species' with v CO at 2000 cm -1 .
A potential of -150 mV was then applied ( Figure S17B). For the native enzyme, this would favour the Ni a -SI state (v CO 1945 cm -1 ), 9,10 which is readily poisoned by CO to give the NiS-CO inhibited state with v CO for the endogenous carbonyl ligand shifted only very slightly to 1944 cm -1 , and a new band for the exogenous, inhibitory CO on Ni(II) at 2054 cm -1 . 9 The R479K variant shows only a minority species with v CO close to 1945 cm -1 when crystals are poised at -150 mV, indicating that Ni a -SI does not accumulate significantly for this variant. Exposing crystals poised at -150 mV to CO results in a change in intensities around 1945 cm -1 (to 1943cm -1 ): it is not possible to assign a band for an exogenous CO, although this could be hidden under the v CN bands. Again, it is clear that there is no absorption band at 2000 cm -1 , and therefore CO exposure at this potential does not give rise to the high wavenumber species.
Poising crystals at +300 mV (under anaerobic conditions, Figure S17C) with no CO exposure gives rise to a spectrum in which the dominant state has a v CO band at 2000 cm -1 and corresponding v CN bands appear at 2110 and 2097 cm -1 . This is consistent with the solution spectrum for anaerobically-oxidised R479K (Figure 13), although the improved signal/noise arising from a high effective protein concentration in the crystals also reveals minor contributions from other states at lower wavenumber.
No change is observed in the spectrum for crystals poised at +300 mV when CO is introduced, confirming that the 'high wavenumber species' is insensitive to CO inhibition. Table S6. Infrared band positions (cm -1 ) of the active site states of selected [NiFe]-hydrogenases in their various active redox states (subscript 'a') and the oxidised inactive state Ni-B. The Ni-SI r state represents an inactive state during the activation of Ni-B to Ni a -SI and is not thought to be involved in the active catalytic cycle. 12 Note that the sub-states of Ni a -L and Ni a -R are presented in descending order, i.e. sub-state I at highest energy, III at lowest as per reference 13 .  [14][15][16][17] ; previously reported Hyd-2 IR bands 10 ; R479K data is presented in Figure 13. Figure S18: Single crystal redox titrations of R479K (oxidative left-hand side, reductive right-hand side) at pH 6.0 between -600 to +400 mV showing the reversible formation of the high wavenumber species with ν CO at 2000 cm -1 at potentials above +148 mV. Data is presented to across an extended wavenumber range (2800-1750 cm -1 ) as raw absorbance data (A) and its second derivative (B) and at a narrower wavenumber range (2200-1750 cm -1 ) as baseline-subtracted (C).

IR spectra of electrochemical redox titrations of R479K single crystals
The microspectroscopic-electrochemical cell 8 was set up anaerobically in a glovebox using H 2 -reduced crystals of R479K in presence of crystal stabilisation buffer (100 mM Bis-tris pH 5.9, 200 mM MgCl 2 , 23% w/v PEG 3350) containing 0.5 mM each of mediators Redox mediator 2,6-dichloroindophenol sodium salt, phenazine methosulfate, indigo carmine, anthraquinone-2-sulfonate, and methyl viologen, and was sealed in the anaerobic environment of the glovebox (<20 ppm O 2 ) before being transferred to the beamline.
Reductive activation of single crystals of R479K in the microspectroscopic-electrochemical cell was performed at -600 mV for until no further changes were observed in the v CO and v CN bands in the IR spectra over a period of approximately 10 min. The oxidative titration was then performed from -600 mV to +400 mV in 25-100 mV steps, only stepping the potential once no further changes were observed in the v CO and v CN bands in the IR spectra over a period of approximately 10 min. The potential was held constant at +400 mV whilst the microscope stage was moved to be able to sample a different area of the crystal for the reductive titration. The potential was then lowered in the reductive direction from +400 to -600 mV at the same potential steps as previously used for the oxidative titration. All aspartic acid residues and glutamic acid residues were modelled as deprotonated except ASP-S-199 which was protonated on the OD2 atom. All lysine and arginine residues were modelled as protonated. Cysteines coordinated with metals, Ni, Fe, and Mg, were modelled as deprotonated. The inorganic residues: [3Fe-4S] cluster, [3Fe-4S] cluster, and the NiFe-cluster were modelled using a nonbonded model. Hirshfeld population analysis of a DFT-CPCM calculation was used to derive atomic charges while CHARMM Lennard-Jones parameters were used for the sulfides.

Theoretical Methods used for Analysis of Hyd-2 R479K Structures
The GROMACS program version 2018.3 6 was used to add missing hydrogen atoms to the model. A cubic box was created with 10 Å between the protein and the edge of the box. Box dimension were 72.09, 78.39, 116.10 Å (length, width, height). The box was filled with 78940 solvent molecules (TIP3 solvent model). The system was neutralized via the addition of 48 Na + ions. The full system size was 267049 atoms. Following system setup, a classical minimization was performed to relax H-atom positions with metal-coordinated waters and waters close to the active site frozen. Classical NVT simulations were performed for 2.5 ns with all metal clusters constrained ([3Fe-4S] cluster, [3Fe-4S] cluster, [NiFe-CO(CN -) 2 X 2 ] and Mg-complex) as well as LYS-479 and the water molecule close to the unknown X 2 ligand bound to Ni. Following the MD simulations, QM/MM as well as QM-cluster models were prepared. For QM/MM geometry optimizations, an active region of 1120 atoms (an approximately 11 Å radius sphere around one of the Ni ion) was defined. Multiple QM-cluster models and QM-regions in the QM/MM calculations were defined and tested. The smallest QM-cluster calculation contained only the NiFe-cluster and the sidechains (SMe groups) of CYS-61, CYS-64, CYS-546, and CYS-549. The smallest QM-region QM/MM model contained in addition to the NiFe-cluster and the 4 cysteine residues, LYS-479 and a water molecule in hydrogen-bonding distance with the diatomic ligand. A large QM-region QM/MM model (150 atoms) was also defined, containing VAL-63, ASP-103, ASP-544, GLU-14, SER-502, HIS-99, HIS-104, and three water molecules, shown in Figure S19.

Vibrational frequency calculations
Harmonic vibrational frequencies were calculated. Both unscaled frequencies as well as frequencies scaled by a factor of 0.9781 are reported. This scaling factor was derived from a calculation of free CO at the same level of theory compared to the experimental frequency.

Calculations of O 2 ligand models.
An O 2 ligand bound to Ni(II) was initially considered. Binding an O 2 ligand to Ni(II) as a terminal ligand lead to spontaneous superoxide (O 2 -) formation in both QM/MM and QM-cluster model calculations, the electronic structure best described as an open-shell Ni(III) ion spin-coupled to an O 2ligand (note: this state is still labelled as Ni(II)-O 2 ). The open-shell singlet state calculated was revealed to be a spincoupled according to analysis of the electronic structure (spin density, Hirshfeld spin populations and Pipek-Mezey localized orbital analysis). This electronic structure was accompanied by bending of the O 2 ligand relative to the Ni ion (a Ni-O-O angle of ~125°), consistent with superoxide formation but clearly inconsistent with the more linear Ni-X-X angle (163°) found in the X-ray structure. Figure S20 and Figure S23 show results for other redox states of the Ni ion (Ni(II), Ni(III), Ni(I)), and multiple spin states of the NiFe cofactor however, all calculated possibilities resulted consistently in a calculated Ni-O-O angle of ~120-126°.

QM/MM results for different ligands bound to R479K
Results of QM/MM calculations with different diatomic ligand possibilities can be found in Figure S20 (calculated Ni-X-X angles), Figure S21 and Table S6 (vibrational frequencies) and Figure S22 (active site heavy-atom distances surrounding the NiFe cofactor).    Figure S21, both unscaled and scaled (using a factor of 0.9781) and additional models from Figure S26. Models are labelled according to formal redox state (assuming metal-based oxidation)

Results of the QM-cluster and QM/MM models with a putative O 2bound ligand
Many calculations (using r 2 SCAN functional) were performed with a putative O 2 ligand, varying the redox state and spin state of the system as shown in Figure S23. The Ni-O-O angle was consistently found to be between 120-130°. Multiple density functional theory methods (r 2 SCAN, TPSS, TPSSh, B3LYP, PBE0 and BHLYP) were also tested for describing the electronic structure of the putative Ni(II)-O 2 model in a broken-symmetry singlet, restricted singlet and triplet states. The functional dependency on the molecular structure is shown in Figure S24; the results reveal that the Ni-Fe distance is rather sensitive to the details of the electronic structure exhibited by each functional (specifically the amount of HF exchange) while the Ni-O-O angle was again found to be consistently between 120-130°. To investigate the effect of the protein environment, QM-cluster models with different surrounding residues included in the model and QM/MM models with different QM-regions were explored for the Ni(II)-O 2 model (see Figure S25). While the Ni-Fe distance showed some small sensitivity with respect to QM-cluster definition or QM-region composition, the Ni-O-O angle was barely affected by the protein environment around the Ni-Fe cluster. Results of models with even more oxidized forms Calculations were also performed on even more oxidized models of the NiFe active site for further exploration of possible near-linear O-O forms or Fe oxidation to Fe(III) which could explain the presence of a strongly shifted CO vibrational mode to ~2000 cm -1 .
i) Further oxidation of the Ni ion beyond the Ni(III)-Fe(II) state would lead to a Ni(IV) ion which could conceivably stabilize a Ni(IV)-peroxo form. We explored this by removing either 1 or 2 electrons from the Ni(II)-Fe(II) redox state with O 2 added as a ligand in an initially linear geometry. As shown in Figure S26, the O 2 ligand remains bent (Ni-O-O angle of ~124°), consistent with superoxide character, as also seen by spin density on the oxygen atoms. The spin density around Ni is consistent with the presence of a S=1/2 Ni(III) ion. Additional spin density could be seen associated with the S-atoms across one or more thiolate ligands suggesting that oxidation has occurred on the S-atoms.
ii) Oxidation of the Fe ion from Fe(II) to Fe(III) could explain a strongly shifted CO frequency. An oxidized NiFe hydrogenase from H. thermoluteolus featuring a Nibound glutamate residue and a bridging cysteine residue (previously Ni-bound) had initially been assigned as a Ni(IV)-Fe(II) state but a recent computational study (Kumar et al. JACS, 2023, 145, 10954-10959) has suggested an antiferromagnetically coupled Ni(III)-Fe(III) state as an alternative possibility. That state also features a strongly shifted CO band at 1993 cm -1 (similar to the experimental 2000 cm -1 band in this work). A Ni(III)-Fe(III) redox state in the O 2 calculations shown in Figure S26 was not revealed as only marginal spin density is associated with the Fe. We further explored the possibility of a Ni(III)-Fe(III) state with a CNligand present (and 2 electrons removed). As the Ni(III)-Fe(III) scenario is likely to arise via antiferromagnetic spin coupling we explored calculations of both a triplet high-spin solution and an antiferromagnetic broken-symmetry singlet solution (found by flipping the spin on either Ni or Fe). Calculations were tested with both the r 2 SCAN functional and the hybrid PBE0 functional (as the HF exchange could help stabilizing spin-coupled solutions). However, as the spin density plots reveal, there is no sign of Fe oxidation in these models either, with only marginal spin density is present around Fe and instead S-based oxidation occurs instead.